Formation of new blood vessels and capillaries (neovascularization) is comprised of two different processes: vasculogenesis, the in situ assembly of capillaries from undifferentiated endothelial cells (EC), and angiogenesis, the sprouting of capillaries from preexisting blood vessels. Vasculogenesis takes place mostly during the early stages of embryogenesis (Folkman and D'Amore, 1996; Yancopoulos et al., 1998). The vasculogenic process can be divided into five consecutive steps (Drake et al., 1998): (1) EC are generated from precursor cells, called angioblasts, in the bone marrow; (2) EC form the vessel primordia and aggregates that establish cell-to-cell contact but have no lumen; (3) a nascent endothelial tube is formed, composed of polarized EC; (4) a primary vascular network is formed from an array of nascent endothelial tubes; and (5) Pericytes and vascular smooth muscle cells are recruited to form the mature vessel.
In mammals, normal angiogenesis is confined to the reproductive system, embryogenesis and development, and repair after injury. Undesirable or pathological neovascularization has been associated with disease states including diabetic retinopathy, psoriasis, cancer, rheumatoid arthritis, atheroma, Kaposi's sarcoma and haemangioma (Fan et al, Trends Pharmacol. Sci. 16: 57-66 (1995); Folkman, Nature Medicine 1: 27-31 (1995)). Alteration of vascular permeability is thought to play a role in both normal and pathological physiological processes (Cullinan-Bove et al, Endocrinology 133: 829-837 (1993); Senger et al, Cancer and Metastasis Reviews. 12: 303-324 (1993)). Intraocular neovascularization is usually associated with diabetic retinopathy and retinopathy of prematurity (King and Brownlee, 1996). The new blood vessels are leaky and rupture easily, which may result in blindness. In chronic inflammatory diseases such as rheumatoid arthritis, new vessels invade the joint surfaces and degrade the cartilage by proteolysis (Battegay, 1995).
But most notably, a growing body of evidence indicates that angiogenesis is essential to the progression of cancer because it is a prerequisite for tumor growth and metastasis (Folkman, 1992). Without vascularization, tumors may remain for years as small (less than a few millimeters) asymptomatic lesions. Weidner et al. New England J. of Med. 324:1-8 (1991). Tumors which become vascularized receive increased oxygen and nutrients through perfusion. Thus, tumors which are vascularized can grow and proliferate. A tumor must constantly stimulate the growth of new capillary blood vessels in order for it to continue to grow. Additionally, angiogenesis allows the tumor cells access to the host animal's circulatory system. The new blood vessels provide a gateway for tumor cells to enter the circulation and metastasize to distant sites. (Folkman, J. Natl. Cancer Inst. 82:4-6 (1990); Klagsbrunn and Soker, Current Biology 3:699-702 (1993); Folkman, J., J. Natl., Cancer Inst. 82:4-6 (1991); Weidner et al., New Engl. J. Med. 324:1-5 (1991)).
In fact, the extent of neovascularity is strongly correlated with metastases in primary breast carcinoma, bladder cancer, prostrate cancer, non-small cell lung cancer, cutaneous melanomas, and uterine cervix carcinoma. (Reviewed in Ferrara, N., Breast Cancer Research and Treatment 36: 127-137 (1995)). In these studies, tumor specimens were histologically analyzed and the number of microvesicles manually counted. The extent of tumor mass vascularization was found to be an independent predictor of the metastatic potential, and more reliable than other prognostic markers.
These results have led researchers to speculate that tumor vascularization could be used as a diagnostic tool to predict metastasis. However, counting of microvesicles in tumor specimens, besides being labor-intensive, is a qualitative art. The method requires considerable technical training in order to obtain reliable and reproducible results. Some groups have reported difficulties in reproducing the method. (Wiedner, N., Amer. J. Path. 147: 9-19 (1995)). Additionally, the process of preparing specimens for histology and counting vesicles is time consuming. Therefore, the application of this technique has been limited generally to research purposes.
Several investigators have theorized that one may be able to measure angiogenic activity in patients by quantitating the presence of angiogenic proteins. There are twelve known angiogenic proteins whose presence could potentially indicate angiogenesis. (Folkman J. New England J. of Med. 333:1757-63 (1995)). Of these factors, those most commonly found to be associated with tumors are basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), insulin growth factor-2, platelet derived growth factor, and colony stimulating factors. Other factors which are candidates for angiogenic and metastatic markers are urokinase-type plasminogen activator and plasminogen activator inhibitor-1, as well as a variety of collagenases and urokinases. (Wiedner, N., Amer. J. Path. 147: 9-19 (1995)).
Recent evidence indicates that VEGF is an important stimulator of both normal and pathological angiogenesis (Jakeman et al, Endocrinology, 133: 848-859 (1993); Kolch et al, Breast Cancer Research and Treatment, 36:139-155 (1995)) and vascular permeability (Connolly et al, J. Biol. Chem. 264: 20017-20024 (1989)). Antagonism of VEGF action by sequestration of VEGF with antibody can result in inhibition of tumor growth (Kim et al, 1993, Nature 362: 841-844). To this end, major progress has been made in the study of molecules that display antiangiogenic activity and may have therapeutic potential. Several naturally occurring antiangiogenic molecules, have been discovered, including: thrombospondin-1, platelet factor-4, fumagillin derivative AGM-1470 (TNP-470), thalidomide, angiostatin and endostatin (Folkman, 1995). These molecules can inhibit endothelial cell (EC) proliferation in vitro, disrupt endothelial tubes, and most importantly, repress tumor growth in vivo (O'Reilly et al., 1997; O'Reilly et al., 1994). Although antiangiogenic factors have attracted much attention, their mechanism of action is not yet clear.
Recent studies have also reported on the presence of circulating endothelial progenitor cells (EPC) in the bloodstream (Asahara et al., 1997; Shi et al., 1998). EPC can be recruited to distinct sites, and upon stimulation with angiogenic factors and cytokines, these cells can differentiate into mature EC and participate in the angiogenic process (Asahara et al., 1999; Takahashi et al., 1999). It has been shown that circulating EPC from human peripheral blood, could be isolated and grown in culture (Asahara et al., 1997). Culturing EPCs in the presence of VEGF enhanced their differentiation to mature EC and promoted the formation of endothelial tubes and subsequent expression of endothelial nitric oxide synthetase. In vivo treatment of mice with VEGF resulted in increasing numbers of circulating EPC (Asahara et al., 1999). Using the cornea micropocket assay, it was shown that VEGF also enhanced the incorporation of EPC into the growing capillaries in the eye.
There thus remains a need for a rapid and objective technique that could be used generally to screen and assess tumor angiogenesis and thus be used (a) to isolate new antiangiogenic agents, (b) to monitor the progress of an antiangiogenic treatment and thereby allow for a custom treatment protocol to be devised, and (c) as a prognostic/diagnostic indicator of angiogenic diseases.